Introduction to AAV Vectors

AAV vectors are developed from single stranded DNA viruses that belong to the Parvoviridae family. This virus is capable of infecting a broad range of host cells, including both dividing and non-dividing cells. In addition, it is a non-pathogenic virus that does not generate an immune response in most of patients.

The AAV genome comprises two open reading frames:

  • Rep: Encode proteins that are required for the AAV life cycle and site-specific integration of the viral genome.
  • Cap: Encode the capsid proteins VP1, VP2 and VP3.

In addition the open reading frames are flanked by inverted terminal repeat (ITR) sequences consisting of 145 bases that have the ability to form a hairpin structure. These sequences are required for the primase-independent synthesis of a second DNA strand and the integration of the viral DNA into the host cell genome.

 

AAV Advantages

Over the last few years, AAV vectors have emerged as an extremely useful and promising mode of gene delivery. This is owing to the following properties of these vectors:

  • AAVs are small, non-enveloped and have only two native genes. Thus can be easily manipulated to develop vectors for different gene therapies.
  • AAV particles are not easily degraded by shear forces, enzymes or solvents. This facilitates easy purification and final formulation of these viral vectors.
  • AAVs are non-pathogenic and have a low immunogenicity. The use of these vectors further reduces the risk of adverse inflammatory reactions. Unlike other viral vectors, such as lentivirus, herpes virus and adenovirus, AAVs are harmless and are not responsible for causing any human disease.
  • Genetic sequences up to 4000 bp (~4 kb) can be delivered into a patient using AAV vectors.
  • Wild type AAV vectors have been shown to insert genetic material in the 19th chromosome of humans. This prevents the chances of ectopic integration of the therapeutic DNA. However, this integrative property is generally eliminated from most AAVs by removing Rep and Cap genes from the viral genome. In such cases the virus remains in an episomal form within the host cells. These episomes remain intact in non-dividing cells, while in dividing cells they are lost during cell division.

 

AAV Limitations

The major drawbacks of these AAV vectors are as follows:

  • The small size of the AAV limits the use of these vectors in cases where a large amount of genetic material is to be delivered. In some cases, the entire genome of the virus has to be replaced with the gene of interest. Researchers are now focussing on increasing the packaging capacity of these vectors by annealing the ITR sequences.
  • The AAV genome must undergo second strand synthesis to produce the double stranded DNA that is required for gene expression. This results in low transduction efficiencies. This limitation can be overcome by packaging the vector as double stranded DNA. However, this reduces the cloning capacity of these vectors